Project Summary/Abstract (30 lines) Variation in treatment response remains a formidable obstacle when selecting ideal therapies for individual patients suffering from various maladies. This problem, in particular, has led to the development of numerous personalized medicine approaches aimed at tailoring therapy on a patient-by-patient basis in order to improve outcomes. Many of these approaches require systematic and quantitative assays to be performed directly on primary cells derived from each patient. However, this poses a particular challenge for translation into the clinic, as the collection, testing, and manipulation of these cells are typically extremely expensive, time- consuming and labor-intensive processes. Rapid improvements in genomic engineering methods have bolstered optimism for the prospect of personalized gene therapy because these methods possess superior modularity, specificity, and capability for rapid correction of disease-conferring genes. However, conventional gene delivery methods continue to require laborious and cumbersome leukapheresis and target-cell purification procedures of patients' blood prior to gene delivery. Moreover, gene engineering suffers from notable shortcomings, such as impermanent inhibition of target functions and unpredictable off-target effects, and demands gene delivery techniques capable of routine and repeated assays on target cells. Efficient multigene delivery methods are thus desirable to reduce off-target toxicity by co-expressing therapeutic and protective markers in therapeutic cells. This project aims to construct a microfluidic primary cell editing platform (pCEP) for robust, affordable, scalable and direct genetic modification of cells purified from bodily fluids. pCEP will selectively trap primary target cells from blood via novel microscale vortices and efficiently co-deliver therapeutic genes and gene editing machinery via automated electroporation of the captured cells. Unlike virus-mediated delivery, pCEP employs a physical gene injection mechanism that offers lower operational costs and higher payloads with the ability to directly deliver Good Manufacturing Practice (GMP)-grade genetic materials. Multiple genes of interest can be sequentially injected into the cytosol in a dose-controlled manner by automated switching of delivery solutions. The versatility and feasibility of the pCEP approach for clinical applications will be validated by performing gene insertion and deletion using model systems for the immortalization of non-proliferating somatic cells via hTERT plasmid injection and production of PD-1 knockout T-lymphocytes via CRISPR-cas9 gene editing, respectively. We envision that pCEP will provide an automated solution for genomic editing of target primary cells directly from bodily fluids as well as a simple and facile means to assess unforeseen adverse effects of newly developed gene-editing techniques for human cells.